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| United States Patent |
6,025,172
|
|
Dani
, et al.
|
February 15, 2000
|
Isolation and sequencing of the hazel FAD2-Ngene
Abstract
The invention relates to the isolation from hazel (Corylus avellana L.) of
the FAD2-N gene coding for the .DELTA.12 desaturase enzyme of the
microsomal fraction and, in particular, provides the nucleotide sequence
and the deduced amino-acid sequence of the gene and provides for the use
of the FAD2-N gene as a probe for the isolation of other plant
desaturases. The invention also relates to the use of the FAD2-N gene for
altering desaturase levels and consequently the fatty-acid composition of
the plant.
| Inventors:
|
Dani; Maria (Sala Di Caserta, IT);
Catello; Sergio (Naples, IT)
|
| Assignee:
|
Soremartec S.A. (BE)
|
| Appl. No.:
|
811177 |
| Filed:
|
March 4, 1997 |
Foreign Application Priority Data
| Current U.S. Class: |
435/189; 435/41; 435/252.3; 435/320.1; 435/325; 435/419; 514/44; 536/23.2 |
| Intern'l Class: |
C12N 009/02; C07H 021/04 |
| Field of Search: |
435/41,189,252.3,320.1,325,419
536/23.2
514/44
|
References Cited
Other References
Okuley et al.; The Plant Cell, vol. 6, 147-158, Jan. 1994; "Arabidopsis
FAD2 Gene Encodes the Enzyme That Is Essential for Polyunsaturated Lipid
Synthesis".
Smith et al.; Nature, vol. 334, 724-726, Aug. 1998; "Antisense RNA
inhibition of polygalacturonase gene expression in transgenic tomatoes".
Flavell; Proc. Natl. Acad. Sci. USA, vol. 91, 3490-3496, Apr. 1994;
"Inactivation of gene expression in plants as a consequence of specific
sequence duplication".
Verwoerd et al.; Nucleic Acids Research, vol. 17, No. 6, 2362, 1989; "A
small-scale procedure for the rapid isolation of plant RNAs".
Valenta et al. "Homology of the major birch-pollen allergen, Bet v I, with
the major pollen allergens of alder, hazel, and hornbeam at the nucleic
acid level as determined by cross-hybridization." Journal of Allergy and
Clinical Immunology (Mar. 1991).
|
Primary Examiner: Wax; Robert A.
Attorney, Agent or Firm: Rothwell, Figg, Ernst & Kurz, P.C.
Claims
What is claimed is:
1. An isolated nucleic acid encoding hazel .DELTA.12 desaturase, wherein
said nucleic acid is DNA encoding the amino acid sequence set forth in SEQ
ID NO. 2.
2. An isolated nucleic acid encoding the .DELTA.12 desaturase according to
claim 1, wherein said .DELTA.12 desaturase comprises the same amino acid
sequence as set forth in SEQ ID NO. 2.
3. An isolated nucleic acid encoding the .DELTA.12 desaturase according to
claim 2, wherein said .DELTA.12 desaturase is of the endoplasmic
reticulum.
4. An isolated nucleic acid from hazel comprising the nucleotide sequence
as set forth in SEQ ID NO. 1 and encoding .DELTA.12 desaturase.
5. An isolated nucleic acid from hazel comprising the nucleotide sequence
as set forth in nucleotides 222-1367 of SEQ ID NO. 1 and encoding
.DELTA.12 desaturase.
6. A substantially purified hazel .DELTA.12 desaturase enyzme of the
endoplamsic reticulum having the amino acid sequence set forth in SEQ ID
NO. 2.
7. A fusion polypeptide comprising the amino acid sequence of the enzyme of
claim 6.
8. An isolated nucleic acid from hazel comprising the "I" clone cDNA (SEQ
ID No: 3).
9. A process for the isolation of genes coding for enzymes having the
function of hazel desaturase or of the desaturase of another species from
genetic material, comprising: (1) hybridizing an isolated nucleic acid
encoding hazel .DELTA.12 desaturase according to claim 1 with said genetic
material; and (2) detecting said genetic material coding for said enzymes.
Description
The present invention relates to the isolation from hazel (Corylus avellana
L.) of the FAD2-N gene which codes for the .DELTA.12 desaturase enzyme of
the microsomal fraction.
More particularly, the invention relates to nucleic acids comprising the
nucleotide sequence of the FAD-N gene, the derived amino-acid sequence of
the gene, and the use as of the gene a probe for the isolation of other
plant desaturases. The invention also relates to the use of this gene for
altering the desaturase levels, and consequently the fatty-acid
composition of the plant. The invention further relates to recombinant DNA
molecules comprising the FAD2-N gene, host organisms comprising these DNA
molecules, the hazel .DELTA.12 desaturase enzyme encoded by the FAD2-N
gene, and fusion polypeptides comprising the enzyme. Host organisms
include vegetable cells, animal cells and micro-organisms. In the fusion
polypeptides of the invention, amino acids added to the polypeptide should
not interfere with the activity of the encoded desaturase, or if so, can
be easily removed from the polypeptide.
Alteration of the fatty-acid composition may have various applications in
the industrial field. One of the greatest problems with hazelnuts is that
they become rancid by oxidation. This is due to the auto-oxidation of
unsaturated lipids with the consequent formation of volatile substances
with a rancid odour which cannot easily be eliminated by the usual
preservation systems. Amongst the possible strategies for reducing the
tendency to become rancid, the best seems to be that of reducing the
degree of unsaturation of the fatty acids present in the kernel oil, since
susceptibility to auto-oxidation is positively correlated with this
parameter. In fact, the rate of peroxide formation is correlated with the
number of C.dbd.C double bonds in the fatty acids. The rate of
auto-oxidation of the fatty acids in comparison with the oleate (18:1) is
about 30 times greater in the linoleate (18:2) and 80 times greater in the
linolenate (18:3). Moreover, the volatile substances resulting from the
degradation of the linoleate and of the linolenate have a lower threshold
of perception than those derived from the oleate. A reduction in linoleic
acid should reduce the availability of substrates for lipoxygenase, reduce
the loss of vitamin E during preservation, and reduce the production of
volatile substances such as hexanals.
In the angiosperms, most of the synthesis of polyunsaturated lipids takes
place by means of a single enzyme, that is, .DELTA.12 (or .omega.6)
desaturase (18:1 desaturase), of the endoplasmic reticulum, although there
is an 18:1 chloroplast desaturase in the leaves of some plants. Moreover,
this enzyme is responsible for more than 90% of the synthesis of
polyunsaturated fatty acids in non-photosynthetic tissues such as, for
example, in the kernels. The conversion of oleic acid (18:1) to linoleic
acid (18:2) thus takes place by means of .DELTA.12 desaturase, and from
linoleic acid to linolenic acid (18:3) by means of .DELTA.15 (or .omega.3)
desaturase.
It has been shown with mutants of Arabidopsis that the FAD2 locus contains
a gene which codes for the oleate desaturase enzyme of the endoplasmic
reticulum (Okuley et al, 1994, The Plant Cell 6, 147-158). The FAD2 gene
was in fact able to complement mutants of Arabidopsis which were deficient
in desaturase activity of the endoplasmic reticulum. The gene coding for
the same enzyme in soya has also recently been isolated and sequenced
(Heppard et al, 1995, Plant Physiol., in press).
A reduction in the .DELTA.12 desaturase levels should therefore lead to a
reduction in the linoleic acid content and, as a secondary effect,
probably also to a reduction in linolenic acid. In hazelnuts the
percentage of linoleic acid varies from 5 to 15%; the percentage of
linolenic acid is from 0.1 to 0.2%. A reduction in these fatty acids
should therefore be useful in the preservation of hazelnuts. There is
therefore clearly a need to isolate the gene which codes for the .DELTA.12
desaturase of the endoplasmic reticulum. The sequence of the gene could
thus be used for gene inactivation in hazelnut kernels. This inactivation
could be carried out either by the antisense technique (Smith et al.
(1988) Nature 334, 724-726) or by the "transwitch" technique (Flavell
(1994) Proc. Natl. Acad. Sci. USA 91, 3490-3496). In the antisense
technique, the hazel would have to be transformed by the entire FAD2-N
gene or by portions thereof, inserted in the opposite direction to the
regulating sequences. In the "transwitch" technique, the hazel would have
to be transformed by an identical copy of the FAD2-N gene. The present
invention also relates to nucleic acids encoding a protein or polypeptide
having an amino acid homology of at least about 80% to the amino acid
sequence of the hazel .DELTA.12 desaturase enzyme of the endoplasmic
reticulum [SEQ ID NO:2] and having the function of the enzyme.
The subjects of the present invention are defined by the following claims.
Embodiments of the present invention will now be described with reference
to the following drawings, in which:
FIG. 1 shows the restriction map of the N2 genome clone,
FIG. 2 (SEQ ID NO:1) shows the nucleotide sequence of the hazel FAD2-N
gene; the amino-acid sequence of the coding portion is also shown (SEQ ID
NO:2);
FIG. 3 (SEQ ID NO:3) shows the nucleotide sequence of the "I" clone of
cDNA,
FIG. 4 shows a comparison between the nucleotide sequences of the "I" (SEQ
ID NO:3) and "N2" (SEQ ID NO:1) clones,
FIG. 5 shows a comparison between the amino acid of the .DELTA.12
desaturases from hazelnut, Arabidopsis and soya,
FIG. 6 shows the homology between hazel .DELTA.12 desaturase and various
desaturases of other plants, both plastid and of the endoplasmic
reticulum,
FIG. 7 shows the expression of the N2 gene in various varieties of hazel,
both in the leaves and in the kernels.
ISOLATION AND CLONING OF THE FAD2 GENE OF ARABIDOPSIS THALIANA FOR USE AS A
PROBE
In order to isolate the gene which codes for hazel .DELTA.12 desaturase
enzyme, it was necessary to use the FAD2 gene of Arabidopsis as a probe.
In order to isolate the Arabidopsis gene, two oligonucleotides were used as
"primers" for the amplification of the sequences included between the
start and the end of the gene. The oligonucleotides used were NOCC1 (SEQ
ID NO:4) (CTGAATTCCAGGTGGAAGAATGCC) which contains the Eco RI restriction
site and the sequences corresponding to the portion between bases 100 and
116 of the gene (Okuley J. et al, 1994, The Plant Cell 6, 147-158) and
NOCC4 (AGGAATTCGACAATTTCTTCACCATCATGC) (SEQ ID NO:5) which contains the
restriction site of the Eco RI enzyme and the sequences complementary to
the portion between base 1245 and base 1266. The amplification reaction
was as follows: 12.8 .mu.l H.sub.2 O, 2.5 .mu.l 10.times.PCR buffer
(Perkin Elmer), 2.5 .mu.l Arabidopsis genome DNA(10 ng/l), 1 .mu.l dNTP,
each 2.5 mM, 2 .mu.l 25 mM MgCl.sub.2, 1 .mu.l NOCC1 oligonucleotide (SEQ
ID NO:4) (50 ng/.mu.l), 1 .mu.l NOCC4 oligonucleotide (SEQ ID NO:5) (50
ng/.mu.l) 0.2 .mu.l Taq I DNA polymerase (Perkin Elmer) (5 U/.mu.l). The
mixture thus prepared was subjected to 1 denaturing cycle for 1 minute at
94.degree. C. and to 40 cycles composed as follows: 30 seconds at
94.degree. C., 1 minute at 52.degree. C., 2 minutes at 72.degree. C. The
amplification products were separated on 1% agarose gel in TAE buffer
(0.04M Tris-acetate, 0.002M EDTA) and stained with ethidium bromide at a
concentration of 0.5 .mu.g/ml. The portion of gel containing the fragment
of the expected length was withdrawn. In order to extract the DNA, 10
.mu.l of Qiaex resin (Qiaex extraction kit, firm Qiagen) were added for
each 200 mg of gel. The supplier's method was then followed. The DNA was
then supplemented with a tenth of a volume of 10.times.H buffer
(Boehringer) and 20 units of Eco RI enzyme (Boehringer). After incubation
overnight at 37.degree. C., the DNA was precipitated with 0.1 volumes of
5M NH.sub.4 OAc and one volume of isopropanol. After 10 minutes at ambient
temperature, the DNA was centrifuged for 20 minutes at 14000 rpm and the
precipitate was washed with 70% ethanol. The DNA was resuspended in 15
.mu.l of H.sub.2 O. The concentration was determined on gel by comparison
with a known standard.
The amplified fragment was inserted in the pUC18 vector. A ligation mixture
was prepared as follows: 1 .mu.l pUC18 plasmid DNA cut with Eco RI (20
ng), 1.5 .mu.l fragment amplified with NOCC1 (SEQ ID NO:4) and 4 (25 ng),
1 .mu.l 10.times. ligase buffer (Boehringer), 1 .mu.l T4 DNA ligase (1
U/.mu.l) (Boehringer), 4.5 .mu.l H.sub.2 O. The reaction mixture was
incubated at 14.degree. C. for 12 hours.
In order to prepare competent cells, the method based on the compound
hexamino-cobalt chloride was used (Maniatis, Molecular cloning, 1989, Cold
Spring Harbor Laboratory Press, 1.76-1.81). 10 .mu.l of the ligation
mixture were added to each aliquot of competent cells, defrosted on ice.
After the cells had been incubated on ice for 30 minutes they were
subjected to thermal shock at 42.degree. C. for 90 seconds and were then
replaced in ice for 60 seconds. After the addition of 0.5 ml of SOC broth
(2% Bactotryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM
MgCl.sub.2, 20 mM glucose, pH7), the cells were incubated at 37.degree. C.
with stirring for 90'. 100, 200 and 300 .mu.l aliquots were spread on
plates containing solid LB broth (10 gr/l NaCl, 10 gr/l Bactotryptone, 5
gr/l yeast extract, pH7.5, 15 gr/l agar) with the addition of 50 .mu.g/ml
of ampicillin and in the presence of IPTG and X-Gal. The plates were then
incubated at 37.degree. C. overnight.
Some of the bacterial colonies obtained were first analyzed for their
plasmid content by a quick method (Maniatis, Molecular cloning, 1989, Cold
Spring Harbor Laboratory Press, 1.32). The colonies containing a plasmid
of the expected length were grown and their plasmid DNA extracted
(Maniatis, Molecular cloning, 1989, Cold Spring Harbor Laboratory Press,
1.33). Those containing a fragment of the expected length (1160 bp) were
identified by digestion of the plasmid DNA with Eco RI. The E1 colony was
selected.
One end of the insert of the E1 colony was sequenced. The plasmid DNA of
the E1 clone was denatured and partially sequenced by Sanger's method
using the enzyme Sequenase and .sup.35 S-dATP (Amersham). The sequencing
products were separated on 8% acrylamide, 8M urea, 1 .times.TBE gel. After
electrophoresis, the gel was dried and exposed overnight in contact with
an autoradiographic plate (.beta. max, Amersham). The sequence was
compared with that published and was identical, identifying the
Arabidopsis FAD2 gene in the cloned fragment.
Extraction of Nucleic Acids from Hazel
Hazelnuts of the Nocchione, Montebello and San Giovanni varieties were
harvested when almost fully ripe. The kernel was skinned before being used
or frozen in liquid nitrogen. The leaves were harvested at a young stage
and frozen in liquid nitrogen. 3 ml of extraction buffer were used for
each gram of vegetable material with the use of the method described by
Verwoerd et al. (Nucl. Ac. Res., 1989, 2362). Upon completion of the
extraction, two selective precipitations were carried out by the addition
of NaCl 2M, and 2 volumes of 95% ethanol to eliminate polysaccharides. The
final pellet was resuspended in H.sub.2 O. Further centrifuging was then
carried out to eliminate any non-resuspended material.
DNA also was extracted from young leaves of the Nocchione and Montebello
varieties. The vegetable tissue was pulverized in liquid nitrogen and the
DNA extracted by the CTAB (REF) method. To eliminate the polysaccharides,
NaCl 2M and 2 volumes of 95% ethanol were added. The samples were
incubated for 15' at -80.degree. C. and centrifuged for 15' at 4.degree.
C. and 14000 RPM (Eppendorf). This selective precipitation was repeated
twice and the final pellet was resuspended in H.sub.2 O. Further
centrifuging was then carried out to eliminate any non-resuspended
material.
Checking of the Probe on Hazel DNA and RNA
About 20 .mu.g of DNA of the Montebello and Nocchione varieties was cut
with Eco RI restriction enzyme in a volume of 300 .mu.l in the presence of
400 units of enzyme and H buffer (Promega), with incubation for one night
at 37.degree. C. After digestion had been checked by gel electrophoresis
of one twentieth of the reaction mixture, the samples were precipitated
with ethanol and resuspended in 30 .mu.l of H.sub.2 O. The DNA was then
subjected to electrophoresis on 0.7% agarose gel and transferred by
capillarity onto nylon membrane (Southern blot) for one night in the
presence of 20.times.SSC (3M NaCl, 0.3M Na citrate). The membrane was
dried in air for 30' and then fixed by UV treatment (120,000
.mu.J/cm.sup.2).
The Arabidopsis .DELTA.12 desaturase gene was used as a probe. For this
purpose, the plasmid DNA of the E1 clone (5 .mu.g) was cut with 20 units
of Eco RI in the presence of H buffer (Boehringer) in a volume of 30 .mu.l
for 12 hours at 37.degree. C. The insert of the clone was separated from
the vector by electrophoresis on 1% agarose gel and extracted from the gel
with the use of Qiaex resin in accordance with the suppliers' instructions
(Qiagen). The DNA was denatured for 10' at 100.degree. C., cooled rapidly
in dry ice, and marked by the random priming method with the use of 6000
Ci/mmol (.alpha..sup.32)P dATP and the reagents of Boehringer's marking
kit.
The nylon membrane containing the hazel DNA was prehybridized for 1.5 hours
at 55.degree. C. in standard buffer (5.times.SSC, 0.1% (w/v)
N-laurylsarcosine, 0.02% SDS, 1% blocking reagent solution) (10% blocking
reagent solution: 10 gr Boehringer blocking reagent in 150 mM NaCl, 100 mM
maleic acid, pH7.5). The membrane was then hybridized with the Arabidopsis
probe for one night at 55.degree. C. The non-hybridized probe was washed
twice for 15' in 2.times.SSC, 0.1% SDS and twice for 15' each in
0.3.times.SSC, 0.1% SDS, always at a temperature of 55.degree. C. The
probe remained coupled to the homologous sequences on the membrane and was
detected by autoradiography.
The RNA extracted from the young leaves of the Montebello and Nocchione
varieties and from the kernels of the San Giovanni variety was separated
on denaturing gel in the presence of formamide and transferred to nylon
membrane by Northern blotting (Maniatis, Molecular cloning, 1989, Cold
Spring Harbor Laboratory Press, 7.43-7.45). 40 .mu.g/sample of total RNA
extracted from San Giovanni kernels, Nocchione leaves and Montebello
leaves were used. 60 pg of probe were used as a positive control. The RNA
was loaded onto a 1% agarose gel in the presence of formaldehyde. The
samples were then subjected to electrophoresis for 3 hours at 80 volts in
the presence of 1.times.MOPS. The gel was rinsed in H.sub.2 O and then
stained with ethidium bromide 0.5 .mu.g/ml to display the RNA. The RNA was
then transferred onto a nylon membrane (Boehringer) by "capillary
blotting" in the presence of 20.times.SSC throughout the night at
4.degree. C. After transfer, the membrane was dried on 3 MM paper and then
fixed by crosslinking using UV light (Stratagene UV Stratalinker 120000
.mu.J/cm.sup.2). The RNA was hybridised with the Arabidopsis .DELTA.12
desaturase probe as described for the DNA. Detection was carried out by
autoradiography. The heterologous Arabidopsis probe was able to display a
band with a molecular weight of about 1500 bp in the hazel RNA and 3 bands
of about 18, 8 and 2.8 kb in the hazel DNA cut with Eco RI.
Construction of a Gene Library of cDNA
The gene library of cDNA was constructed from RNA from kernels harvested
when almost fully ripe and taken from plants of the San Giovanni variety.
For this purpose, the Poly(A)+mRNA was isolated from the total RNA with
the use of the Poly(A)Tract mRNA Isolation System II, in accordance with
the method provided by the firm Promega. The samples were eluted in
H.sub.2 O and precipitated with 0.1 volumes of 3M NaOAc and 3 volumes of
95% ethanol. After one night at -80.degree. C., the RNA was centrifuged
for 15' at 14000 rpm (Eppendorf), the pellet was rinsed in 75% ethanol and
resuspended in 10 .mu.l of H.sub.2 O. The concentration was read with a
spectrophotometer and the yield was 3.2 .mu.g of Poly(A)+mRNA per mg of
total RNA.
The messenger RNA polyadenilate derived from kernels of the San Giovanni
variety was used as a template for the synthesis of complementary DNA
(cDNA) with the use of Boehringer's "cDNA synthesis kit" in accordance
with the method recommended by the suppliers. An extraction was then
carried out with one volume of phenol:chloroform:isoamyl alcohol
(25:24:1). The cDNA was then purified in a Pharmacia column (cDNA spun
columns) after the addition of NaCl 100 mM. The buffer used was the
following: 10 mM Tris-HCl pH 7.5, 1 mM EDTA, 150 mM NaCl. Eco RI
"adaptors" (Pharmacia) were added to the ends of the cDNA. The reaction
mixture contained: 5 .mu.l of cDNA (half of the cDNA obtained from 6 .mu.g
of Poly(A)+RNA), 10 .mu.l of ligase buffer 10.times.(Promega), 10 .mu.l of
Eco RI adaptors (0.01 u/.mu.l), 6 units of T4 DNA ligase (Promega), in a
final volume of 100 .mu.l. After incubation for 12 hours at 12.degree. C.,
the ligase enzyme was inactivated for 10' at 65.degree. C. Phosphorylation
of the adaptors then followed by the addition, to the 100 .mu.l mixture,
of 10 .mu.l of 100 mM ATP and 10 units of T4 polynucleotide kinase. After
incubation at 37.degree. C. for 30', the enzyme was inactivated by
incubation for 10' at 65.degree. C. Purification was then carried out with
one volume of phenol:chloroform:isoamyl alcohol (25:24:1). The cDNA was
then purified from fragments of less than 400 bp as follows. After the
addition of NaCl to a final concentration of 0.1M NaCl, the cDNA was
separated by chromatography in a column with Sepharose CL-4B resin (Size
prep 400 spun column, Pharmacia) according to the method suggested by the
suppliers. The fragments of cDNA shorter than 400 bases were thus
excluded. The cDNA was precipitated with one thirtieth of a volume of 3M
NaOAc and 2 volumes of 95% ethanol, centrifuged and resuspended in 10
.mu.l of H.sub.2 O.
The cDNA was inserted in the .lambda. phage vector Zap II cut with Eco RI
and dephosphorylated (Stratagene) in the following manner: 2 .mu.l of cDNA
(200 ng), 1 .mu.l of .lambda. Zap II cut with Eco RI (1 .mu.g/.mu.l)
(Stratagene), 0.5 .mu.l of T4 DNA Ligase (4 U/.mu.l) (Promega), 0.5 .mu.l
of 10.times.ligation buffer (Promega), 1 .mu.l of H.sub.2 O. The reaction
mixture was incubated for 14 hours at 12.degree. C. The mixture containing
the cDNA inserted in the vector was used for the reconstruction of the
phages with the use of Stratagene's Gigapack Gold "in vitro packaging"
kit. The gene library of phages thus obtained was constituted by about
300,000 pfu (plaque-forming units). In order to amplify the gene library,
XL1 Blue MRF' cells were prepared as described by Stratagene and used the
same day. The gene libraries were plated at a concentration of about 5000
pfu per plate (95 cm.sup.2). After growth, the phages were resuspended in
SM (5.8 gr/l NaCl, 2 gr/l MgSO.sub.4.7H.sub.2 O , 50 ml/l 1M Tris HCl (pH
7.5), 5 ml/l 2% gelatine) and, after the addition of chloroform to 5% and
incubation for 15 minutes at ambient temperature, the cell debris was
centrifuged for 10 minutes at 2000.times.g. Chloroform to 0.3% was added
to the supernatant liquid and the phages were preserved at 4.degree. C.
Aliquots were preserved at -70.degree. C. after the addition of DMSO to
7%. The gene library was titled.
Construction of a Partial Genome Gene Library
The DNA of the Nocchione variety was digested with Eco RI restriction
enzyme and separated on agarose gel. The fragments with lengths of up to
10000 bp (base pairs) were isolated from the gel with the use of Qiaex
resin according to the Qiagen's method. For cloning in the .lambda. vector
Zap II, 400 ng of DNA fragments were incubated with 1 .mu.g of
desphosphorylated .lambda. Zap II (Stratagene) in the presence of ligase
buffer and 1.5 units of T4 DNA ligase (Promega) for 12 hours at 14.degree.
C.
Strategene's Gigapack Gold "in vitro packaging" kit was used in accordance
with the suppliers' instructions to make up the gene library. The gene
library of phages thus produced was amplified as described for the cDNA
gene library. The complexity of the gene library was 1,500,000 clones.
This gene library was also amplified.
Screening of the cDNA Gene Library
About 250,000 phages of the cDNA gene library were plated on LB broth in
the presence of XL1 Blue MRF' cells, divided into 12 plates each
containing 20,000 pfu. After growth, the phages were transferred onto
nylon membranes and their denatured DNA was fixed on the membranes as
described by Boehringer for screening with non-radioactive probes. The
membranes were then hybridized with the Arabidopsis .DELTA.12 desaturase
gene. The probe was prepared by the isolation of the insert containing the
entire coding region of the gene from the plasmid. The insert was then
marked with digoxigenin-dUTP with the use of Boehringer's "DNA labelling
kit". Prehybridization was carried out in standard buffer (Boehringer) and
hybridization was carried out in the same buffer with the addition of the
Arabidopsis probe at a concentration of 10 ng/ml and at a temperature of
55.degree. C.
After washing twice in 2.times.SSC, 0.1% SDS for 5 minutes at ambient
temperature and washing twice in 0.3.times.SSC, 0.1%SDS at 55.degree. C.,
detection was carried out with the use of an anti-digoxigenin antibody
conjugated with alkaline phosphatase (Boehringer) and a chemiluminescent
substrate (AMPPD, Boehringer).
11 positive phage plaques were identified. These were isolated, the phages
resuspended in SM and titled. From 50 to 200 phages were plated for each
positive plaque. The plaques were transferred onto nylon membranes and
subjected to a second hybridization with the Arabidopsis .DELTA.12
desaturase probe, as already described above. The following clones which
could hybridize with the Arabidopsis .DELTA.12 desaturase gene were
obtained from the second screening: I, F, 4.
Screening of the Genome Gene Library
The gene library of Nocchione DNA was subjected to screening in the same
way as the cDNA gene library. 1,600,000 phages were plated, divided into
40 plates. After growth, they were transferred to nylon membranes as
described for the cDNA gene library. The membranes were then hybridized
with the Arabidopsis .DELTA.12 desaturase gene as described for the cDNA
gene library. Autoradiography of the membranes showed 9 positive plaques.
These plaques were isolated, titled and subjected to a second screening. 6
plaques were re-confirmed as positive. 4 of these gave a very strong
signal.
Analysis of the Clones Isolated
The following positive phage clones were converted into plasmids by in vivo
excision in accordance with the method suggested by Stratagene (Gigapack
Gold in vitro packaging): I, F, 4 (cDNA gene library), N2, N11, N17, N18,
N21, N25 (genome gene library).
The plasmid DNA of the clones of the cDNA gene library was isolated and the
length of the insert analyzed by digestion with Eco RI. The plasmid DNA of
the genome clones was isolated, the length of the insert analyzed by
cutting with restriction enzyme, and the clones rechecked by hybridization
with the Arabidopsis probe. FIG. 1 shows the map of the N2 genome clone.
Sequencing
The N2 clone was selected from the genome clones. For sequencing, the
insert was fragmented with Sau3A restriction enzyme and the fragments
obtained were subcloned in pUC18 vector cut with BamHI (Maniatis,
Molecular cloning, 1989, Cold Spring Harbor Laboratory Press, 1.68-1.69).
The clones obtained were analyzed both for the length of the insert and by
hybridization with the Arabidopsis probe. Since the N2 insert was 2.8 kb
and hence longer than the .DELTA.12 desaturase gene, the hybridization
excluded the clones containing sequences outside the gene. The insert of
the I, F, 4 and N2 clones was isolated and sequenced with the use of the
Sequenase kit and (35S)dATP. All of the clones (cDNA and genome) were
first sequenced at the ends with the use of primers which could couple
with the vector in both orientations. In order to complete the internal
regions and to assemble the fragments of the N2 genome clone, internal
oligonucleotides were then designed and synthesized and were used for the
sequencing. The following table shows the sequences of the internal
oligonucleotides:
______________________________________
OLIGONUCLEOTIDE SEQUENCE
______________________________________
N2-3SS (SEQ ID NO:6)
CAG ACC AGC ATC CGA GAC
- N2-3SD (SEQ ID NO:7) GGA TTG GCT TAG GGG GGC
- N2-29R'S (SEQ ID NO:8) GCC AAC CAT GTC ATC AAC CC
- NOCCS (SEQ ID NO:9) ATG GTA GAG AAG AGA TGG TG
- COL (SEQ ID NO:10) CTG GTG GGT TGT TGA AG
- N2-S1N (SEQ ID NO:11) GGA GAG GTC ATA AAC AAC
______________________________________
The I and F clones were sequenced entirely. As far as the N2 clone is
concerned, only the regions corresponding to the FAD2-N gene were
sequenced. FIGS. 2 (SEQ ID NO:1) and 3 (SEQ ID NO:8) show their sequences.
The I and F cDNA clones were identical. A comparison between the I (SEQ ID
NO:3) and the N2 (SEQ ID NO:1) genome clone showed the same sequence (FIG.
4), indicating that N2 (SEQ ID NO:1) contains the gene which codes for the
cDNA of the I clone (SEQ ID NO:3).
Comparison Between the Gene Isolated and Other Desaturases
The nucleotide and amino-acid sequence of the N2 clone (SEQ ID NO:1) was
compared with other desaturases (FIG. 6). The greatest homology was with
the two .DELTA.12 desaturases of the endoplasmic reticulum of soya and
Arabidopsis and with a hydroxylase of ricin which uses the same substrate
as .DELTA.12 desaturase. Homology with the plastid .DELTA.12 desaturases
and with both the plastid and endoplasmic reticulum .DELTA.15 desaturases
was, however, much lower. FIG. 5 shows the comparison between the amino
acid sequence of hazel .DELTA.12 desaturase and those sequences for
.DELTA.12 desaturase of Arabidopsis and soya.
Checking the Expression of the Hazel .DELTA.12 Desaturase Gene
RNA was extracted from kernels of the San Giovanni, Montebello and
Nocchione varieties and from leaves of the Montebello and Nocchione
varieties. After separation on agarose gel, the RNA was transferred onto a
nylon membrane and hybridized with the insert of the I clone marked with
digoxigenin. The result is shown in FIG. 7, in which a band is visible in
the kernel RNA but not in that of the leaves.
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# SEQUENCE LISTING
- - - - (1) GENERAL INFORMATION:
- - (iii) NUMBER OF SEQUENCES: 11
- - - - (2) INFORMATION FOR SEQ ID NO:1:
- - (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1662 base - #pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
- - (ii) MOLECULE TYPE: Genomic DNA
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
- - CCTCATAAAA AAGTAAGCTC ATTTACCTCA AGTAGGGTTT CCTTATGACA AA -
#TGAGTCCC 60
- - GCAATCCTTT TCTATGAGGT GCTATAATTG CAAATGTCCA AATCATAGGG AT -
#ATGGATCC 120
- - AAATACTATT AATATTATGT AGTGTGTTTT TTTTTTTCCC TCAAATTTAC TC -
#TCACACCT 180
- - AAGTTGATTT TCTCCAGCAT TGGACATAGC CTCTGTAGAC AATGGGAGCT AG -
#AAGCCGAA 240
- - TGCCTGCTAC CAACAAGCCT AAAGAGCAAA AAACACCCAT CCAGCGAGCA CC -
#ACACACAA 300
- - AACCCCCATT CACTCTTAGC CAACTCAAGA AAGCCGTCCC ACCCAATTGT TT -
#CCAACGCT 360
- - CTCTCCTACG CTCGTTCTCA TATGTTGTTT ATGACCTCTC CTTAGCCTTC CT -
#CTTCTACT 420
- - ATATTGCTAC CTCTTACTTC CATCTCCTCC CTCACCCCCT TTCCTACTTG GC -
#ATGGTCAA 480
- - TCTATTGGGC TCTCCAAGGC TGCATTCTCA CCGGCGTTTG GGTCATCGCA CA -
#TGAGTGCG 540
- - GTCACCATGC CTTTAGTGAC TACCAATGGG TTGATGACAT GGTTGGCCTA AC -
#CCTTCACT 600
- - CTGCTCTTTT AGTTCCATAC TTTTCATGGA AGATTAGCCA CTGTCGCCAC CA -
#CTCTAACA 660
- - CCGGCTCCCT TGACCGAGAT GAGGTGTTTG TCCCCAAGCC GAAATCCAAA AT -
#GCCATGGT 720
- - TTTCTAAGTA CTTCAACAAC CCACCAGGTA GGGTCCTCAC TCTTTTGATC AC -
#ACTCACTC 780
- - TAGGCTGGCC CTTGTACTTA GCCTTGAATG TTTCTGGCCG ACCCTATGAT CG -
#TTTTGCTT 840
- - GCCACTATGA TCCCTATGGC CCCATTTATT CCAATCGCGA AAGGTGTCAA AT -
#ATTTGTCT 900
- - CGGATGCTGG TGTCTTTGCT ACAACTTATG TGCTTTACTA CGCAGCAATG TC -
#AAAAGGGC 960
- - TGGCATGGCT TGTATTCATT TATGGTATGC CATTGCTCAT AGTGAATGGC TT -
#CCTTGTAT 1020
- - TAATCACCTA CTTGCAGCAC ACTCACCCTG CATTGCCGCA CTATGACTCA TC -
#GAATGGGA 1080
- - TTGGCTTAGG GGGGCATTGG CGACGGCGGA TAGAGATTAC GGAATGCTGA AT -
#AAGGTTTT 1140
- - CCACCAATAT CATAGACACC CATGTGGCTC ACCATCTCTT CTCTACCATG CC -
#TCATTACC 1200
- - ATGCAATGGA AGCCACCAAA GCAATCAAGT CAATATTGGG CAAATACTAC CA -
#GTTTGATG 1260
- - GCACTCCAGT TTACAAGGCA GTGTGGAGGG AGGCTAAAGA GTGCCTTTAT GT -
#TGAGTCGG 1320
- - ACGAGGGGGC CCCTAACAAA GGTGTTTTCT GGTATCAGAG CAAGCTGTGA TA -
#TTGGCTGG 1380
- - ATAGAGCCAA AGAAAATGTG ATTAGTAAGG TAGTGTCTTT GGTCAGTTTG GT -
#GTGTTAAG 1440
- - GAACAAATAA TAATAATTAG CGACTATGAA TAGTTATTGT TAAACAAAAT TC -
#ACCCTTAT 1500
- - GTTTAGCAGG AACTTTTCTG GCTACACTTT TTTTCGTATG AAAAGCGCAT AT -
#TTTTTAAT 1560
- - TGTTATATTG TTTTGACATT ACTCAAGCTT CAAAATTAAT ATCACAGAAA AT -
#ATCCAATG 1620
- - TCGAAGGTTT CATTGTAGGT TGAAAACTTT ATATTGAGGT GG - #
- #1662
- - - - (2) INFORMATION FOR SEQ ID NO:2:
- - (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 382 amino - #acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
- - Met Gly Ala Arg Ser Arg Met Pro Ala Thr As - #n Lys Pro Lys Glu Gln
1 5 - # 10 - # 15
- - Lys Thr Pro Ile Gln Arg Ala Pro His Thr Ly - #s Pro Pro Phe Thr Leu
20 - # 25 - # 30
- - Ser Gln Leu Lys Lys Ala Val Pro Pro Asn Cy - #s Phe Gln Arg Ser Leu
35 - # 40 - # 45
- - Leu Arg Ser Phe Ser Tyr Val Val Tyr Asp Le - #u Ser Leu Ala Phe Leu
50 - # 55 - # 60
- - Phe Tyr Tyr Ile Ala Thr Ser Tyr Phe His Le - #u Leu Pro His Pro Leu
65 - #70 - #75 - #80
- - Ser Tyr Leu Ala Trp Ser Ile Tyr Trp Ala Le - #u Gln Gly Cys Ile Leu
85 - # 90 - # 95
- - Thr Gly Val Trp Val Ile Ala His Glu Cys Gl - #y His His Ala Phe Ser
100 - # 105 - # 110
- - Asp Tyr Gln Trp Val Asp Asp Met Val Gly Ly - #s Thr Leu His Ser Ala
115 - # 120 - # 125
- - Leu Leu Val Pro Tyr Phe Ser Trp Lys Ile Se - #r His Cys Arg His His
130 - # 135 - # 140
- - Ser Asn Thr Gly Ser Leu Asp Arg Asp Glu Va - #l Phe Val Pro Lys Pro
145 1 - #50 1 - #55 1 -
#60
- - Lys Ser Lys Met Pro Trp Phe Ser Lys Tyr Ph - #e Asn Asn Pro Pro
Gly
165 - # 170 - # 175
- - Arg Val Leu Thr Leu Leu Ile Thr Leu Thr Le - #u Gly Trp Pro Leu Tyr
180 - # 185 - # 190
- - Leu Ala Leu Asn Val Ser Gly Arg Pro Tyr As - #p Arg Phe Ala Cys His
195 - # 200 - # 205
- - Tyr Asp Pro Tyr Gly Pro Ile Tyr Ser Asn Ar - #g Glu Arg Cys Gln Ile
210 - # 215 - # 220
- - Phe Val Ser Asp Ala Gly Val Phe Ala Thr Th - #r Tyr Val Leu Tyr Tyr
225 2 - #30 2 - #35 2 -
#40
- - Ala Ala Met Ser Lys Gly Leu Ala Trp Leu Va - #l Phe Ile Tyr Gly
Met
245 - # 250 - # 255
- - Pro Leu Leu Ile Val Asn Gly Phe Leu Val Le - #u Ile Thr Tyr Leu Gln
260 - # 265 - # 270
- - His Thr His Pro Ala Leu Pro His Tyr Asp Se - #r Ser Glu Trp Asp Trp
275 - # 280 - # 285
- - Leu Arg Gly Ala Leu Ala Thr Ala Asp Arg As - #p Tyr Gly Met Leu Asn
290 - # 295 - # 300
- - Lys Val Phe His Asn Ile Ile Asp Thr His Va - #l Ala His His Leu Phe
305 3 - #10 3 - #15 3 -
#20
- - Ser Thr Met Pro His Tyr His Ala Met Glu Al - #a Thr Lys Ala Ile
Lys
325 - # 330 - # 335
- - Ser Ile Leu Gly Lys Tyr Tyr Gln Phe Asp Gl - #y Thr Pro Val Tyr Lys
340 - # 345 - # 350
- - Ala Val Trp Arg Glu Ala Lys Glu Cys Leu Ty - #r Val Glu Ser Asp Glu
355 - # 360 - # 365
- - Gly Ala Pro Asn Lys Gly Val Phe Trp Tyr Gl - #n Ser Lys Leu
370 - # 375 - # 380
- - - - (2) INFORMATION FOR SEQ ID NO:3:
- - (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1133 base - #pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- - (ii) MOLECULE TYPE: cDNA to mRNA
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
- - TCCAACGCTC TCTCCTACGC TCGTTCTCAT ATGTTGTTTA TGACCTCTCC TT -
#AGCCTTCC 60
- - TCTTCTACTA TATTGCTACC TCTTACTTCC ATCTCCTCCC TCACCCCCTT TC -
#CTACTTGG 120
- - CATGGTCAAT CTATTGGGCT CTCCAAGGCT GCATTCTCAC CGGCGTTTGG GT -
#CATCGCAC 180
- - ATGAGTGCGG TCACCATGCC TTTAGTGACT ACCAATGGGT TGATGACATG GT -
#TGGCCTAA 240
- - CCCTTCACTC TGCTCTTTTA GTTCCATACT TTTCATGGAA GATTAGCCAC TG -
#TCGCCACC 300
- - ACTCTAACAC CGGCTCCCTT GACCGAGATG AGGTGTTTGT CCCCAAGCCG AA -
#ATCCAAAA 360
- - TGCCATGGTT TTCTAAGTAC TTCAACAACC CACCAGGTAG GGTCCTCACT CT -
#TTTGATCA 420
- - CACTCACTCT AGGCTGGCCC TTGTACTTAG CCTTGAATGT TTCTGGCCGA CC -
#CTATGATC 480
- - GTTTTGCTTG CCACTATGAT CCCTATGGCC CCATTTATTC CAATCGCGAA AG -
#GTGTCAAA 540
- - TATTTGTCTC GGATGCTGGT GTCTTTGCTA CAACTTATGT GCTTTACTAC GC -
#AGCAATGT 600
- - CAAAAGGGCT GGCATGGCTT GTATTCATTT ATGGTATGCC ATTGCTCATA GT -
#GAATGGCT 660
- - TCCTTGTATT AATCACCTAC TTGCAGCACA CTCACCCTGC ATTGCCGCAC TA -
#TGACTCAT 720
- - CAGAATGGGA TTGGCTTAGG GGGGCATTGG CGACGGCGGA TAGAGATTAC GG -
#AATGCTGA 780
- - ATAAGGTTTT CCACAATATC ATAGACACCC ATGTGGCTCA CCATCTCTTC TC -
#TACCATGC 840
- - CTCATTACCA TGCAATGGAA GCCACCAAAG CAATCAAGTC AATATTGGGC AA -
#ATACTACC 900
- - AGTTTGATGG CACTCCAGTT TACAAGGCAG TGTGGAGGGA GGCTAAAGAG TG -
#CCTTTATG 960
- - TTGAGTCGGA CGAGGGGGCC CCTAACAAAG GTGTTTTCTG GTATCAGAGC AA -
#GCTGTGAT 1020
- - ATTGGCTGGA TAGAGCCAAA GAAAATGTGA TTAGTAAGGT AGTGTCTTTG GT -
#CAGTTTGG 1080
- - TGTGTTAAGG AACAAATAAT AATAATTAGC GACTATGAAT AGTTATTGTT AA - #A
1133
- - - - (2) INFORMATION FOR SEQ ID NO:4:
- - (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base - #pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
- - CTGAATTCCA GGTGGAAGAA TGCC - # - #
24
- - - - (2) INFORMATION FOR SEQ ID NO:5:
- - (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base - #pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
- - AGGAATTCGA CAATTTCTTC ACCATCATGC - # - #
30
- - - - (2) INFORMATION FOR SEQ ID NO:6:
- - (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base - #pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
- - CAGACCAGCA TCCGAGAC - # - #
- # 18
- - - - (2) INFORMATION FOR SEQ ID NO:7:
- - (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base - #pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
- - GGATTGGCTT AGGGGGGC - # - #
- # 18
- - - - (2) INFORMATION FOR SEQ ID NO:8:
- - (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base - #pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
- - GCCAACCATG TCATCAACCC - # - #
- # 20
- - - - (2) INFORMATION FOR SEQ ID NO:9:
- - (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base - #pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
- - ATGGTAGAGA AGAGATGGTG - # - #
- # 20
- - - - (2) INFORMATION FOR SEQ ID NO:10:
- - (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base - #pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
- - CTGGTGGGTT GTTGAAG - # - #
- # 17
- - - - (2) INFORMATION FOR SEQ ID NO:11:
- - (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base - #pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
- - GGAGAGGTCA TAAACAAC - # - #
- # 18
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